Conversion of alpha-hydroxyalkylated residues in biomolecules using methyltransferases

ABSTRACT

The present invention relates to targeted conversion of alpha-hydroxyalkylated residues in biomolecules in the presence of a directing methyltransferase, namely to targeted removal of the alpha-hydroxyalkyl moieties to give unmodified residues, or targeted derivatization of the alpha-hydroxyalkyl groups by covalent coupling of non-cofactor compounds represented by formula HQ-LX1 wherein X represents a functional group or a reporter group attached via a linker moiety L, and QH is selected from HS—, HSe—, HO—H 2N —, HN 3  or HCN in the presence of a directing methyltransferase. Further development of the method of targeted conversion comprises methods for targeted labeling a biomolecule and method for detecting hydroxymethylated target sites in a biomolecule according to the present invention.

FIELD OF THE INVENTION

The present invention relates to methyltransferase-directedsequence-specific covalent conversion of alpha-hydroxyalkylated residuesin modified biomolecules, namely relates to i)methyltransferase-directed removal of alpha-hydroxyalkyl groups to yieldunmodified residues, and ii) methyltransferase-directed derivatizationof alpha-hydroxyalkyl groups by coupling non-cofactor nucleophiliccompounds in said modified biomolecule.

Namely, this invention covers a method for targeted conversion ofalpha-hydroxyalkylated target residues in biomolecules as well as amethod for targeted labeling of alpha-hydroxyalkylated target residuesin biomolecules, method for detecting alpha-hydroxyalkylated targetresidues in a biomolecule and a kit for performing the methodsmentioned, all grounded on the idea of conversion ofalpha-hydroxyalkylated target residues using a cofactor-free directingmethyltransferase.

The present invention is exemplified using DNA methyltransferases(MTases). However, it can also be used with RNA methyltransferases aswell as methyltransferases acting on other biomolecules.

In this description the term “methyltransferase” refers to enzymes thatnormally transfer the methyl from S-adenosyl-L-methionine (AdoMet) ontotheir substrate. Preferably, the methyltransferase is an enzyme capableof methylating DNA, RNA or (poly)peptides. More preferably, themethyltransferase is a DNA cytosine-5 methyltransferase that uses acovalent activation mechanism for the transfer of the methyl groups onthe C5 position of a target cytosine residue. More preferably, themethyltransferase is selected from M.HhaI, M.SssI, M.HpaII or aderivative thereof. The term “M.HhaI” refers to the DNAmethyltransferase deposited in the Swissprot database under accessionnumber P05102. All MTases used in this invention are cofactor-free, i.e.MTase preparations contain no more than 2 mol % of bound endogenouscofactor AdoMet.

The term “biomolecule” means DNA, RNA or (poly)peptide. The term“(poly)peptide” refers alternatively to peptide or to polypeptide.Preferably, the biomolecule is chromosomal or genomic DNA. Biomoleculesmay be entirely natural, i.e. unmodified, synthetic or modified and mayexist as complexes. For example the term “nucleic acid molecule”comprises DNA and RNA molecules or RNA/DNA hydrids as well as modifiedDNA and RNA molecules. DNA may be for example cDNA or genomic DNA. RNAmay be for example mRNA, hnRNA, tRNA, rRNA etc.

The term “modified biomolecule” means a biomolecule that containsmodified residues. Modified residues are those that contain additionalchemical entities (modifying side chains) as compared to normal majorcomponents of the biomolecule. DNA normally consists of four majorunmodified residues (C, T, A, G); a certain fraction of C and A residuesare found to be modified in natural DNA. Such modifications aretypically brought about via action of intracellular enzymes (see below).

The term “conversion of a modified residue” means either removal of themodifying moiety to give an unmodified residue, or furtherderivatization (chemical extension) of that moiety to give a derivatizedresidue.

The term “removal” means chemical splitting of a compound by breaking astable covalent bond (such as a C—C or N—C bond) thus yielding anunmodified residue and a small molecule, typically an aldehyde.

The term “derivatization” means extending a biomolecule by coupling ofchemical entities, such as carbon chains, chemically reactive groups orreporter groups into biomolecules, such as DNA without otherwisechanging the target biomolecule.

The term “coupling” means chemical addition of a compound by making astable covalent bond (such as a C—C bond, C—O bond, C—S bond, C—Se bondor C—N bond). The coupling reaction can be an addition of an entireexogenous compound to a target biomolecule or its condensation whereby ahydroxyl group in the target biomolecule is replaced with a wholemolecule of the exogenous compound with release of a water molecule(H₂O).

BACKGROUND OF THE INVENTION

Besides the four major nucleobases (C, A, G and T), DNA of most livingorganisms contains minor amounts of their methylated variants:5-methylcytosine (5mC), N4-methylcytosine and N6-methyladenine. Thesemethylated species are formed by DNA methyltransferase enzymes (MTases),which catalyze the transfer of a methyl group from the cofactorS-adenosyl-L-methionine (AdoMet) to form the above methylatednucleotides at specific positions of their target sequences (Cheng,(1995) Annu. Rev. Biophys. Biomol. Struct. 24, 293-318). It is wellestablished that DNA methylation is an important biological mechanismthat regulates gene expression in vertebrate animals including humans(Bird, A. (2002) Genes Dev. 16, 6-21), Goll, M. G. & Bestor, T. H. Annu.Rev. Biochem. 74, 481-514 (2005) and serves as a species self-code inbacteria. Genomic DNA sometimes contains 5-hydroxymethylated pyrimidinenucleobases 5-hydroxymethylcytosine and 5-hydroxymethyluracil (hmC andhmU) (Gommers-Ampt, J. H. & Borst, P. (1995) FASEB J. 9, 1034-1042).

Glucosylation of the 5-hydroxymethyl groups in certain bacteriophagesand an African trypanosome serves to protect the invading genome againsthost defense systems. The presence of hmC was previously reported in DNAfrom animal brains (Penn et al., (1972) Biochem. J. 126, 781-790).Recent studies of genomic DNA from human neurons and brains(Kriaucionis, S. & Heintz, N. Science 324, 929-930), as well as DNA frommouse embryonic stem cells (Tahiliani, M. et al. Science 324, 930-935)found that hmC residues occur at CG sequences and that they are likelyproduced by oxidation of mC residues. The 5-hydroxymethyl groups in DNAmay alter interactions with cellular proteins involved in epigeneticcontrol of gene activity (Valinluck, V. et al. (2004) Nucleic Acids Res.32, 4100-4108), whereas elevated levels of hmU in DNA were reported tocorrelate with incidents of breast cancer (Djuric, Z. et al. (1996)Cancer 77, 691-696). Altogether, the above evidence suggests that5-hydroxymethylate nucleobases, and hmC in particular, may playimportant roles in embryonic development, brain function and cancerprogression. However, neither the chromosomal localization of hmCresidues nor the underlying biological mechanisms are currently known,and further studies are required to address these fundamental issues.Most importantly, such studies are hampered by the lack of adequateanalytical techniques that would permit facile analysis of hmC residuesin DNA.

Current analytical techniques to study cytosine modifications inmammalian DNA are based on the existence of two epigenetic states ofcytosine in CG sites: unmodified cytosine (C) and 5-methylated cytosine(5mC). To this end, numerous techniques have been developed for theidentification and localization of 5mC in DNA (Schumacher et al. (2006)Nucleic Acids Res. 34, 528-542). The gold standard method to study thegenomic localization of individual 5mC residues is bisulfite sequencing(Frommer et al. (1992) Proc Natl Acad Sci USA 89, 1827-1831) and itsnumerous modifications. This method is based on bisulfite-mediateddeamination of C to U; 5mC residues are inert to this reaction, andtherefore standard sequencing of bisulfite-converted DNA shows 5mCresidues in the C-track, whereas T and C residues—in the T-track. Iftreated with bisulfite, hmC is converted to cytosine 5-methylsulfonate,which is deaminated at an even slower rate than 5mC (Hayatsu, M. &Shiragami, M. (1979) Biochemistry 18, 632), and should thus appear inthe C-track. Therefore, hmC residues cannot be distinguished from mCresidues using conventional bisulfite sequencing protocols. Similarly,other high-throughput genome-wide analysis techniques such as mDiP(methylated DNA immunoprecipitation) (Weber et al. (2005) Hum Mol Genet.14, R11-R18), which is based on binding m5C-containing DNA fragments to5mC-specific antibodies, or methods based on using methylation sensitiverestriction endonucleases, are not suitable for detection of hmCresidues either. Therefore, as all the existing techniques were designedto distinguish only the two alternate states of cytosine (methylatedversus unmodified) (Schumacher et al. (2006) Nucleic Acids Res. 34,528-542), they have none or poor ability to determine hmC residues ingenomic DNA.

Recently, a method for DNA derivatization using non-cofactor reactionsof DNA methyltransferases has been disclosed (patent applicationLT2009023 filed Feb. 4, 2009). This technique permitsmethyltransferase-directed sequence-specific covalent coupling offormaldehyde (or other aliphatic aldehydes) to the C5-position of theirtarget cytosine residues in DNA, thereby producing 5-hydroxymethylated(or 5-hydroxyalkylated-) cytosines. The application also describesmethods for subsequent sequence-specific covalent derivatization of hmCresidues in various types of DNA molecules by methyltransferase-directedcoupling of nucleophilic compounds, including thiols. The latterreaction in principle permits derivatization of hmC residues in DNA withvarious functional and reporter groups provided that they occur at atarget position for the directing MTase. Since hmC residues are known tooccur at CG sequences in genomic DNA of vertebrate animals includinghumans, some of the derivatization reactions may be useful in developingthe required techniques for analysis of hmC in DNA. However, thesederivatization reactions have not been assessed with respect to theirsuitability for chemical manipulation and analysis of hmC residues invarious types of DNAs, including mammalian genomic DNA.

In conclusion, it is obvious that new reliable and validated methods arerequired for analysis of hmC resides in genomic DNA.

SUMMARY OF THE INVENTION

This problem can be overcome by providing the embodiments characterizedin claims 1-14 of the present invention.

The key idea of the present invention relates to the targeted conversionof hydroxyalkylated residues in a biomolecule in the presence of adirecting methyltransferase. Such hydroxyalkylated residues in abiomolecule, that are the target residues of a directingmethyltransferase, were found by the authors to be selectively convertedto unmodified residues or to derivatized residues in the presence of themethyltransferase. One example of substrate-activatingmethyltransferases is pyrimidine-5 specific methyltransferases, whichnaturally catalyze the transfer of a methyl group to the 5 position ofcytosine or uracil residues in DNA, RNA or free nucleotides by making atransient covalent bond to the 6 position of the pyrimidine ring.

The present invention covers a use of a cofactor-free methyltransferasefor targeted conversion of a modified biomolecule, bearing a modifyingmoiety of formula —CH(OH)—R, wherein R is hydrogen or C₁-C₁₂-alkyl,preferably hydrogen or lower alkyl, into an unmodified biomolecule byremoval of said modifying moiety at the methyltransferase target site.

The present invention also covers a method for targeted conversion of amodified biomolecule, comprising incubation of the modified biomolecule,bearing a modifying moiety of formula —CH(OH)—R, wherein R is hydrogenor C₁-C₁₂-alkyl, preferably hydrogen or lower alkyl, with acofactor-free directing methyltransferase under conditions compatiblewith enzymatic activity of the methyltransferase, wherein said targetedconversion results from: i) covalent removal of said modifying moiety atthe target site; or ii) derivatization of said modifying moiety at thetarget site by covalent coupling of non-cofactor nucleophiliccompound(s) of general formula HQ-LX, wherein X represents a functionalgroup or a reporter group attached via a linker L, and Q is selectedfrom S, Se, O, N, C.

In the preferred embodiments of present invention R is hydrogen or —CH₃and Q is S or Se. The biomolecule is a nucleic acid molecule, preferablyDNA. Said methyltransferase is a DNA cytosine-5 methyltransferase,selected from the group consisting of M.HhaI, M.SssI, and M.HpaII orderivatives thereof.

The modified biomolecule used in the present invention is a naturally orartificially modified biomolecule, bearing a modifying moiety mentionedabove at the target residue.

The method according to present invention also applies to a method fortargeted labeling of a modified biomolecule, comprising a direct orsubsequent incorporation of a reporter group, which is suitable as alabel and which allows for the identification of the labeled moleculeamong other unlabeled molecules.

The present invention also relates to a method for detectinghydroxymethylated target sites in a biomolecule, comprisingderivatization or labeling of the biomolecule by coupling non-cofactornucleophilic compounds of present invention in the presence of acofactor-free methyltransferase and detecting whether the target sitesof said methyltransferase have been modified, wherein modification ofthe target site of said methyltransferase is indicative of the presenceof hydroxymethylated target site. The method of present inventionextends to the cases, wherein the coupled compound(s) is added to a5-hydroxymethylcytosine residue in DNA and cannot be added to a5-methylcytosine or a cytosine residue.

Finally, the present invention relates to a kit comprising acofactor-free directing methyltransferase or a cofactor-free directingmethyltransferase and an non-cofactor nucleophilic compound(s), suitablebuffer components and further comprising aldehyde scavenging compoundsfor performing any of the methods above.

DESCRIPTION OF DRAWINGS

To illustrate the main characteristic features of the present inventionthis description contains:

FIG. 1: Reversed-phase HPLC analysis of enzymatically fragmented duplexoligodeoxynucleotides obtained after treatment with M.HhaI.hmC-containing DNA duplex I:II (13 μM) was treated with M.HhaI (15 μM)for 2 hours at 37° C. (trace 2). Control reaction (trace 1) lackedM.HhaI. Boxed area contains peaks corresponding to dC and hmdC.

FIG. 2: TLC analysis of [³³P]-labeled 2′-deoxy-5′-mononucleotides ofenzymatically fragmented duplex oligodeoxynucleotides after treatmentwith M.HhaI, M.HpaII and M.SssI.

20 nM hmC-containing cognate oligodeoxynucleotide duplex was treatedwith catalytically active or C81S mutant M.HhaI (lanes 2, 3), withcatalytically active or thermally inactivated M.HpaII (lanes 5, 6) orcatalytically active or thermally inactivated M.SssI (lanes 8, 9) for 1hour at room temperature.

FIG. 3: TLC analysis of [³³P]-labeled 2′-deoxy-5′-mononucleotides ofenzymatically fragmented duplex oligodeoxynucleotides after treatmentwith cysteine or selenocysteine in the presence of M.HhaI. 20 nMoligodeoxynucleotide duplexes containing C, 5mC, hmC at the targetresidues position of the GCGC site or an C or hmC residue in the CCGGsite as shown all were treated with M.HhaI (lanes 1-6), and 50 mML-cysteine (lanes 1-5) or 1 mM L-selenocysteine (lane 6) for 1 hour atroom temperature.

FIG. 4: Restriction endonuclease analysis of methyltransferase-directedmodification of a 618 bp 5-hydroxylalkylcytosine-containing DNAfragment. The DNA fragment (100 nM) containing hmC (left panel) or heC(right panel) residues at the target GCGC site was incubated with M.HhaI(wild type or C81S mutant as shown) for 2 hours at 37° C. Modified DNAswere fragmented with a restriction endonuclease R.Hin6I and analyzed byagarose gel electrophoresis.

FIG. 5: Restriction endonuclease analysis of methyltransferase-directedmodification of a 618 bp 5-hydroxylalkylcytosine-containing DNAfragment. The DNA fragment (100 nM) containing hmC residues at thetarget CG sites was incubated with M.SssI (lanes 1, 2) or HpaII (lanes3, 4) for 2 hours at 37° C. MTase treated DNAs (lanes 2 and 4) anduntreated controls (lanes 1 and 3) were fragmented with a restrictionendonuclease R.Hin6I (lanes 1, 2) or R.HpaII (lanes 3, 4) and analyzedby agarose gel electrophoresis.

FIG. 6: TLC analysis of [³³P]-labeled 2′-deoxy-5′-mononucleotides ofenzymatically fragmented genomic DNA after treatment with M.SssI. Humangenomic DNA was treated with M.SssI for 2 hours at 37° C. and thencleaved with R.HpaII or R.MspI endonucleases as indicated in the rightbottom corner, ³³P-labeled at its 5′-ends, digested to5′-mononucleotides and analyzed by TLC (lane 3). Control samples (lanes1 and 2) were not treated with M.SssI, and analyzed as above.Densitometric scans of the image (lane 1—dotted line; lane 2—gray line;lane 3—solid thin line) are shown on the right. The arrow points at theposition corresponding to the hmC nucleotide in the density scans.

FIG. 7: Gel electrophoretic analysis of methyltransferase-directedmodification of a 618 bp 5-hydroxylalkylcytosine-containing DNAfragment. The DNA fragment (100 nM) containing hmC residues at thetarget CG sites was incubated with 1,4-dithiothreitol in the presence ofM.HhaI or M.SssI and then treated with a Oregon Green maleimide. LabeledDNA was analyzed by 2% agarose gel electrophoresis. Imaging of thefluorescein reporter was performed using a 473 nm laser scanner (leftpanel), DNA fragments were visualized after staining with ethidiumbromide (right panel). Lane 2, M.SssI; lane 3, M.HhaI; lane 1, controlwith MTase omitted.

FIG. 8: Reversed-phase HPLC analysis of enzymatically fragmented duplexoligodeoxynucleotides obtained after treatment with M.HhaI, compound (I)and compound (II). 13 μM DNA duplex I:II was incubated, in the presenceof 15 μM M.HhaI, with 13 mM formaldehyde for 40 min (trace 2) and thenadding compound (II) to a final concentration of 300 mM2-mercaptoethanol (trace 3), 50 mM L-cysteine (trace 4), 400 μM5′-deoxy-5′-thioadenosine (trace 5), 12 mM cysteamine (trace 6), 50 mMdithiothreitol (trace 7), 1 mM selenocysteine (trace 8), 1.2 mMselenocysteamine (trace 9) or 50 mM hydroxylamine (trace 10) andincubating for 1 hour at room temperature. Control reaction (trace 1)lacked M.HhaI. The HPLC elution buffer A was 20 mM ammonium acetate pH5.5. Arrows point at peaks corresponding to the new modificationproducts.

DETAILED DESCRIPTION OF THE INVENTION

The major scope of the present invention is the use of amethyltransferase for targeted conversion of alpha-hydroxyalkylatedresidues in a biomolecule by covalent detachment or extension of thealpha-hydroxyalkyl side chain in a target residue of the biomolecule.

The general principle of targeted conversion of alpha-hydroxyalkylatedresidues in a biomolecule according to the present invention can beunderstood from the Scheme 1 below which shows possibilities ofconversion of 5-alpha-hydroxyalkylcytosine residues in DNA in thepresence of DNA cytosine-5 methyltransferases (MTase).

Scheme 1. Sequence specific conversion of alpha-hydroxyalkylatedresidues in DNA with DNA methyltransferases according to the presentinvention. Reaction 1: targeted removal of alpha-hydroxyalkyl groupsfrom target cytosine residues in DNA; Reaction 2: targetedderivatization of alpha-hydroxyalkyl groups in target cytosine residuesin DNA.

In support of the novelty and inventive step of the present invention itshould be noted:

1) the reaction of removal of alpha-hydroxyalkyl groups from cytosineresidues described in the present invention (Reaction 1 in Scheme 1) isnovel and is not obvious to the skilled person. The described reactionis atypical for MTases, which naturally catalyze cofactor-dependenttargeted transmethylation (nucleophilic substitution S_(N)2), includingreactions with AdoMet analogs.2) targeted removal 5-alpha-hydroxyalkyl groups including5-hydroxymethyl groups from cytosine residues DNA (Reaction 1 inScheme 1) cannot be achieved by any means described in the prior art.3) derivatization of 5-alpha-hydroxyalkyl cytosine residues (includinghmC) in DNA by targeted coupling of nucleophilic compounds (Reaction 2in Scheme 1) has been described previously as a second step of atwo-step procedure for sequence-specific derivatization of unmodifiedDNA (LT2009023). In the present invention it is shown that naturallyoccurring hmC residues in DNA can also be derivatized and then labeledin a similar manner.

In a preferred embodiment of the present invention, R comprises H and—CH₃, and -QH comprises —SH and —SeH. However it is obvious to theperson skilled in the art that R might be easily extended to C₁-C₁₂alkyl, alkenyl, alkynyl, and -QH to cover at least —OH, —NH₂, —NHNH₂ or—ONH₂, N₃H, NCH (or corresponding salts in which a hydrogen atom isreplaced by a cation, such as a metal ion or an ammonium ion) as well asother suitable nucleophiles that are sufficiently active in aqueousbuffers in the pH range of 4-10. In preferred embodiments of the presentinvention, L is —CH₂CH(CO₂H)—, but it is understandable to the skilledperson that L is also covering another suitable linker groups rangingfrom just a mere covalent bond to a combination of linear, cyclic and/oraromatic moieties optionally connected with —NHCO—, —O—, —S— connectors,(poly)ethyleneglycol chains —(CH₂CH₂O)_(n)— n=1-100, etc.

Treatment of 5-hydroxyalkylcytosine-containing DNA with an exogenousnucleophilic compound HQ-LX (wherein LX comprises a chemical reactivegroup or a reporter group X attached via a linker L) in the presence ofa directing DNA cytosine-5 methyltransferase permits sequence-specificcoupling of the compound to give incorporation of the LX group via athiomethyl anchor (when QH═—SH) at the target cytosine residues. Thechemical reactive group X can then be used for covalent ligation with asuitable compound carrying a reporter group (see below).

When QH═SeH, a similar work out leads to targeted incorporation of theLX group via a selenomethyl anchor into DNA, as demonstrated in Examples2 and 5. The chemical reactive group X can then be used for covalentligation with a suitable compound carrying a reporter group. Selenides,which are part of the selenomethyl anchor, can be readily oxidized toselenoxides by treatment with H₂O₂ or NalO₄. Since selenoxides cansubsequently undergo elimination with the cleavage of a Se—C bond(Wirth, T. (2000) Angew. Chem. Int. Ed. 39, 3740-3749; Gieselman et al.(2002) ChemBioChem 3, 709-716), the selenomethyl anchor can thus be usedas a chemically cleavable covalent linker in affinity purifications oflabeled biomolecules. On the other hand, the presence of a Se atom inthe attached group can be used for applications such as crystallographicdetermination of biomolecular structure via multi-wavelength anomalousdispersion (MAD) technique due to significant anomalous scattering ofX-rays. The presence of the ⁷⁷Se nucleus (natural abundance of 8%) witha S=½ magnetic spin may also find applications in NMR and EPRspectroscopy (Zelakiewicz et al. J. Am. Chem. Soc., 2004, 126,8112-8113).

Further development of the proposed method for targeted conversion ofhmC residues in DNA is a method for targeted labeling a biomoleculecomprising modification (derivatization) of the biomolecule according toLT2009023 and, accompanied by incorporation of a group that is suitableas a label and that allows for the identification of the labeledbiomolecules among other unlabeled molecules.

In a preferred embodiment of the present invention sequence-specificlabeling of DNA was achieved by DNA methyltransferase-directed couplingof L-cysteine (thiol) followed by chemo-selective ligation with an e.g.affinity label, such as amine-reactive biotin.

Scheme 2. Selective sequence-specific covalent labeling of hmC residuesin DNA using DNA methyltransferases according to the present invention.The attached reporter moiety is shown as a ball.

Scheme 2 above demonstrates the principle of such sequence-specificlabeling of hmC-containing genomic DNA achieved by (1) treatment ofhmC-containing DNA with L-cysteine in the presence of the HhaImethyltransferase, followed by (2) amino-selective ligation of areporter moiety with an N-hydroxy-succinimide ester.

In the preferred embodiment of the present invention, (a) thenoncofactor nucleophilic compound or a subsequent derivative thereofcontains a fluorescent label; and (b) hydroxymethylated targets sitesare detected by the presence of fluorescence in said nucleic acidmolecule.

In a preferred embodiment of the present invention sequence-specificfluorescent labeling of DNA was achieved by DNAmethyltransferase-directed coupling of 1,4-dithiothreitol followed bychemo-selective ligation with an e.g. affinity label, such asthiol-reactive fluorophore (Example 13).

In another preferred embodiment of the present invention, the label ofsaid detectable compound is detected by (a) an antibody specificallybinding to the label of said detectable compound or by (b) avidin orstreptavidin specifically binding to the label of said detectablecompound.

As mentioned, numerous ways to achieve targeted labeling or targetedderivatization of biopolymers can be realized according to presentinvention. For this purpose a noncofactor nucleophilic compound shouldtypically contain a chemical moiety LX, wherein X comprises a functionalgroup or a reporter group that is attached via a linker group L.

Many chemo-selective ligations defining the reactive groups X areavailable for attaching a label to the modified biomolecule in aqueoussolution. Classical ligations (Garman, (1997) Non-radioactive labeling:A practical introduction, Academic Press) involve primary amino groupswhich can be reacted with amine reactive groups likeN-hydroxy-succinimidyl ester, acyl azide, acyl nitrile, acyl chloride,pentafluorophenyl ester, thioester, sulfonyl chloride, isothiocyanate,imidoester, aldehyde or ketone leading to stable amides, sulfonamides,thioureas, imidates or imines, which can be reduced to stable secondaryamines. Thiols specifically react with haloacetamides, maleimides,aziridines or other thiols leading to thioether or disulfide linkagesand 1,2-diols can be modified with arylboronic acids. Hydrazines orhydroxylamines can be condensed with aldehydes or ketones leading tohydrazones or oximes. 1,2-Aminothiols selectively react with aldehydesor thioesters to form thiazolidines (e.g. N-terminal cysteine residuesof polypeptides, Liu and Tam, (1994) Proc. Natl. Acad. Sci. USA 91,6584-6588) or stable amide bonds (e.g. N-terminal cysteine residues ofpolypeptides, native chemical peptide ligation, Dawson et al., (1994)Science 266, 776-779); azides can be reacted with alkynes (Huisgen1,3-dipolar cycloaddition, Lewis et al. (2002), Angew. Chem. Int. Ed.41, 1053-1057) or with phosphane esters (Staudinger ligation, Saxon andBertozzi, (2000) Science 287, 2007-2010) to form 1,2,3-triazoles oramides; Diels-Alder cycloadditions between activated dienes anddienophiles (e.g. furanes and maleimides, Graham et al., (2002) Tet.Lett. 4785-4788) are feasible in aqueous solution. Other modernpalladium-catalyzed cross-coupling reactions between arylhalides andterminal alkynes (Sonogashira coupling, Casalnuova and Calabrese, (1990)J. Am. Chem. Soc. 112, 4324-4330; Dibowski and Schmidtchen, (1998)Angew. Chem. Int. Ed. 37, 476-478; Bong and Ghaderi, (2001) Org. Lett.3, 2509-2511) or between arylhalides and arylboronic acids (Suzukicoupling, Casalnuova and Calabrese, (1990) J. Am. Chem. Soc. 112,4324-4330; DeVasher et al., (2004) J. Org. Chem. 69, 7919-7927) yieldingarylalkynes or biaryls could be used. Additionally, copper-catalyzedalkyne coupling reactions between terminal haloalkynes and terminalalkynes or terminal silylalkyne leading to conjugated diynes can beperformed in aqueous solution. Finally, fluorogenic derivatizationreagents like 4-halo-7-nitrobenzofurazan, N-methylisatoic anhydride oractivated bimanes can be used to label transferred thiol, amino orhydroxyl groups directly.

Nucleic acids generally do not contain highly nucleophilic orelectrophilic centers. Thus, besides the cycloadditions,palladium-catalyzed cross-coupling reactions or copper-catalyzed alkynecoupling reactions, many other reactions between nucleophiles andelectrophiles with interchangeable reactive group X could be used forsequence-specific labeling of nucleic acids.

In the preferred embodiment of the present invention X comprises atleast one functional group, selected from a primary amino group, a thiolgroup, a 1,2-diol group, a haloacetamide group, a maleimide group, analdehyde group, a ketone group, an azido group, an alkyne group, a1,3-diene function, a dienophilic function, an arylhalide group, aterminal alkyne group, an arylboronic acid group, a terminal haloalkynegroup, a terminal silylalkyne group and a protected amino, thiol,1,2-diol, hydrazino, hydroxyamino, aldehyde, ketone and 1,2-aminothiolgroup. For the purposes of labeling of biopolymers X also comprisesheavy atoms or heavy atom clusters suitable for phasing of X-raydiffraction data, radioactive or stable rare isotopes, and a residue ofa member selected from fluorophores, fluorescence quenchers,chromophores, affinity tags, spin labels (stable paramagnetic groups),groups containing radioactive or stable rare isotopes, groups containingheavy atoms suitable for phasing X-ray diffraction data, crosslinkingagents, nucleic acids cleaving groups, haptens, nanoparticles and beads.

Two major strategies could be used to identify hmC residues in genomicDNA (Scheme 3).

A. One strategy is based on selective conversion of hmC residues tounmodified cytosine residues. This approach is can be used with existinganalysis methods that normally can reveal positions of 5mC andunmodified C in DNA, but do not distinguish hmC from 5mC. For example,bisulfite sequencing will show the positions of 5mC+hmC residues in theC-track and positions of unmodified cytosines in the T-track. SelectiveMtase-directed conversion of hmC to C and subsequent bisulfitesequencing the converted DNA would then reveal positions of 5mC residuesin the C-track, and positions of hmC+C residues in the T-track.Comparison of the two datasets will display hmC residues as bands thatmigrated from the C-track to the T-track upon enzymatic conversion.

B. Another strategy is based on selective derivatization of hmC residuessuch that they can be directly distinguished from 5mC and C residues.For example, selective coupling of thiols or selenols in the presence ofdirecting DNA Mtases will lead to corresponding thiomethyl orselenomethyl derivatives. Such derivatives can be used to attachreporter or affinity groups (see Scheme 2).

Scheme 3. Sequence specific conversions of natural DNA with DNAmethyltransferases according to the present invention for analyticaldetermination of hmC residues. A. conversion of hmC residues tounmodified cytosines; B. derivatization/labeling of hmC residues.

In yet another preferred embodiment of the present invention, (a) theattached moiety interferes with nucleic acid amplification at therecognition sites of the methyltransferase; and (b) hydroxymethylatedtarget sites are detected by testing whether amplification of thenucleic acid molecule at the recognition sites of the methyltransferasehas been retarded. Retardation of amplification may be achieved byinterfering with primer binding or with strand elongation during anamplification reaction.

During the methods of present invention, a step of nucleic acidsequencing may be performed. Any methods known in the art may be usedfor sequencing.

In a preferred embodiment of the present invention, PCR is real-timePCR. In another preferred embodiment of the present invention, nucleicacid amplification is carried out by real-time PCR.

In another preferred embodiment of the present invention, (a) nucleicacid molecules modified at the methyltransferase recognition sequenceare purified by affinity purification; and (b) the compounds of formulas(I) used in the present invention or a derivative thereof contains anaffinity tag.

In preferred embodiments, the methods of the present invention compriseafter the step of targeted modification of a biomolecule an additionalstep of sequencing the DNA molecule. Any methods known in the art may beused for sequencing.

Examples provided in the present invention (Example 3, FIG. 3) show thatan exogenous nucleophilic compound or a derivative thereof is added to a5-hydroxymethylcytosine residue and cannot be added to a5-methylcytosine residue or cytosine residues in DNA.

In yet another preferred embodiment of the present invention, theidentity of said DNA molecule is determined by DNA sequencing,hybridization, MALDI-TOF or analysis of nucleoside composition byenzymatic fragmentation and chromatography.

Finally, in one of the preferred embodiments, the kit of the presentinvention comprises a methyltransferase or methytransferase and anon-cofactor nucleophilic compound in separate containers and mayfurther contain an information leaflet or instruction for use.

Scavenging compounds can be a further component of such a kit, suppliedto sequester the aldehyde released from the reaction such that topreclude it from entering the reverse reaction. All classes of watersoluble compounds that react with aldehydes (thiols, primary amines,hydroxylamines, hydrazines) can be used. These compounds can also besupplied in a chemically altered form such as modified with a protectinggroup, containing a sterically demanding group (to prevent theirreactivity as active nucleophiles with respect to the biomolecule), inan oligomeric or polymeric form that releases the compound(s) whenbrought into a suitable milieu such as a methyltransferase buffer, orimmobilized to a polymer or a bead. For example thiols exist in anoxidized form as disulfides or polysulfides etc., which easily convertto thiols under reducing conditions. Auxiliary biomolecules containingthe above functional groups (for example proteins such as bovinealbumin, etc) can also be as scavenging agents provided that theycontain no target residues for the directing MTase.

In another preferred embodiment the present invention also relates to akit containing a methyltransferase and/or diagnostic formulation(s) onthe basis of above. In one of the embodiments of present invention thediagnostic formulation is a liquid composition. The preferred solvent ofthe diagnostic formulation is aqueous in nature. In addition, theformulation may contain other ingredients or carriers for modifying ormaintaining the pH, osmolarity, viscosity, clarity, color, sterility,stability, rate of dissolution, or odor of the formulation. Similarly,the formulation may contain still other pharmacologically acceptableingredients for modifying or maintaining the stability, rate ofdissolution, release, or absorption of the diagnostic composition. Oncethe diagnostic formulation has been formulated, it may be stored insterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin ready to use form or requiring reconstitution immediately prior touse.

In practice for the preparation of an unmodified biomolecule accordingto the present invention the following steps are to be carried out:

a) combining (putting together) the modified biomolecule and acofactor-free MTase in a suitable aqueous buffer that is compatible withenzymatic activity of the methyltransferase (for example: 50 mM MOPS, 50mM MES pH 7.5, 1 mM Na₂EDTA, 15 mM NaCl, 0.2 mg/ml bovine serum albumin,5% glycerol; or 10 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5 mM Na₂EDTA 0.2mg/ml bovine serum albumin, 5% glycerol; or other similar bufferrecommended by a MTase manufacturer);b) incubation of the reaction at a temperature that is compatible withenzymatic activity of the methyltransferase (as recommended by a MTasemanufacturer) for a period of 5-120 min;c) stopping the reaction (by adding an inhibiting compound, diluting thereaction with a suitable solvent, flash-freezing at −20° C. or lowertemperature, or inactivating the methyltransferase by heating at 40° C.above the optimal reaction temperature for 5-20 min);d) isolation of the unmodified biomolecule as necessary.

For the preparation of a derivatized biomolecule according to thepresent invention the following steps are to be carried out:

a) combining (putting together) the biomolecule, a cofactor-free MTaseand a non-cofactor nucleophilic compound in a suitable aqueous buffer(see above);b) incubation of the reaction at a temperature that is compatible withenzymatic activity of the methyltransferase for a period of 5-120 min;c) stopping the reaction (see above);d) isolation of the unmodified biomolecule as necessary.

Normally, directing MTases are supplied at near equimolar amounts withrespect to biomolecular target sites. A non-cofactor nucleophiliccompound is typically supplied at a millimolar concentration.

EMBODIMENTS OF THE INVENTION

Represented below are specific examples of the embodiments of thepresent invention. The scope of the invention is only illustrated bythese examples without being restricted to them.

Examples 1-2

Sequence-specific modifications of hmC-Containingoligodeoxyribonucleotide duplexes with the HhaI DNA cytosine-C5methyltransferase.

Modification was first performed in the presence of the HhaI DNAcytosine-05 methyltransferase (M.HhaI) using short duplexoligodeoxyribonucleotides. M.HhaI recognizes the 5′-GCGC-3′ target sitein DNA and naturally transfers the methyl group ofS-adenosyl-L-methionine (SAM or AdoMet) to the C5 position of the innercytosine residue (underlined). Following the enzymatic modificationreactions, a duplex oligodeoxynucleotide was enzymatically fragmented to2′-deoxynucleosides and analyzed by reversed-phase HPLC coupled withESI-MS.

The duplex oligodeoxynucleotide I:II was produced by annealingsingle-stranded oligonucleotide I (SEQ ID NO:1)(5′-TAATAATGCGCTAATAATAATAAT) and II (SEQ ID NO:2)(3′-TTATTACGCGATTATTATTATTA) in water as described in LT2009023. hmCmodification was introduced enzymatically as described in LT2009023.Modification removal reactions were performed by incubating thehmC-modified duplex oligonucleotide I:II (13 μM) with M.HhaI (15 μM) for2 hour at 37° C. Derivatization reactions were performed by incubatingthe modified duplex oligonucleotide I:II (13 μM) with 1 mML-selenocysteine in the presence of M.HhaI (15 μM) for 1 hour at 20° C.For nucleoside composition analysis DNA was isolated as described inLT2009023 and treated with Nuclease P1 (2 u, Sigma, Germany) for 2 hoursat 60° C. and calf intestine alkaline phosphatase (30 u, Fermentas LifeSciences, Lithuania) overnight at 37° C. Obtained nucleosides wereanalyzed by reverse-phase HPLC (Discovery C18 75×2.1 mm, 3 μm column,equipped with a Supelguard Discovery C18 20×2.1 mm, 5 μm precolumn,Supelco, Germany) coupled with a mass spectrometric detector (HP 1100series ESI-MS equipped with singe quadruple). Compounds were eluted witha linear gradient of solvents A (20 mM ammonium acetate pH 5.5) and B(80% aqueous methanol) at a flow of 0.3 ml/min at 30° C. as follows:0-20 min, 0-20% B; 20-22 min, 20-100% B; 22-27 min, 100% B. Analyteswere detected by an in-line diode array UV absorbance detector. UVabsorbance spectra were acquired (190-400 nm wavelength interval) atpeak maxima and solvent contributions were removed by subtractingbackground spectra before and after the peaks. For online massspectrometric detection post-column mobile phase modification (equalco-flow of 96% methanol, 4% formic acid and 1 mM sodium hydroxide) wasused to enhance the detection efficiency of 2′-deoxycytidine and itsderivatives. Mass spectra were recorded in 50-600 m/z range in thepositive ion mode. Ionization capillary voltage was 5000 V, fragmentervoltage was 100-120 V, drying gas temperature was 300-350° C. and flowrate was 10-12 L/min. High-resolution mass spectra (HR-MS) were acquiredby analysis of corresponding HPLC fractions on a LTQ Orbitrap massspectrometer (Thermo Electron) equipped with a Proxeon NanoSpray ESI.

Example 1

Nucleoside composition analysis of a hmC-containing duplexoligodeoxyribonucleotide after treatment with M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) revealed, besides dG, dT and dAnucleosides, the presence of dC and dC^(CH) ² ^(OH) (hmC2′-deoxynucleoside) with a retention times of 3.7 and 4.2 min,respectively (see FIG. 1). These compounds were analyzed by coupledESI-MS (m/z: 250 [M+Na]⁺, 134 [cytosine+Na]⁺ and m/z: 280 [M+Na]⁺, 164[5-hydroxymethylcytosine+Na]⁺). The observed masses are in agreementwith 2′-deoxycytidine dC and 5-hydroxymethyl-2′-deoxycytidine dC^(CH) ²^(OH), respectively. The latter compound was nearly fully converted tounmodified dC upon incubation with M.HhaI (compare the relative size ofC and hmC peaks in traces 1 and 2). Thus the hydroxymethyl group isremoved from the 5-hydroxymethylcytosine residue in DNA by M.HhaI.

Examples 2-6

Sequence-specific modifications of internally labeled hmC-containingoligodeoxyribonucleotide duplexes with a DNA cytosine-05methyltransferase DNA modification was investigated in the presence of adirecting DNA cytosine-5 MTase M.HhaI, M.SssI, or M.HpaII. M.HhaIrecognizes the double-stranded DNA sequence 5′-GCGC-3′ and naturallytransfers the methyl group of S-adenosyl-L-methionine (SAM or AdoMet) tothe C5 position of the inner cytosine residue (underlined). Accordingly,other DNA methyltransferases perform a similar reaction but theyrecognize different DNA target sequences: M.SssI (recognition targetCG), or M.HpaII (recognition target CCGG). Analysis of modifications ofa target cytosine residue in the presence of a DNA cytosine-C5methyltransferase was performed using internally labeled duplexoligodeoxyribonucleotides. Internally labeled duplexoligodeoxyribonucleotides contained a cognate target sequence of a DNAmethyltransferase in which a target cytosine residue (or a targetresidue and another cytosine residue for M.HhaI) were ³³P-labeled. hmCmodification was introduced enzymatically by incubation of the duplexoligodeoxynucleotide with formaldehyde (13 mM) and a correspondingMTase.

For composition analysis, a duplex oligodeoxynucleotide wasenzymatically fragmented to 2′-deoxynucleoside-5′-monophosphates andthen analyzed by TLC and autoradiography permitting selectiveobservation of modifications of the target nucleotide.

The duplex oligodeoxyribonucleotides III:IV (unmethylated duplex forM.HhaI), V:VI (hemimethylated duplex for M.HhaI), VII:VIII (duplex forM.HpaII and M.SssI) were produced by mixing equal molar amounts (150 μM)of complementary single-stranded oligodeoxyribonucleotide III (SEQ IDNO:3) (5′-TCGGATGTTGTGGGTCA) and IV (SEQ ID NO:4)(3′-GCCTACAACACCCAGTCGCGTACTATCACAT); V (5′-TCGGATGTTGTGGGTCAG) (SEQ IDNO:5) and VI (SEQ ID NO:6) (3′-GCCTACAACACCCAGTCGMGTACTATCACAT); VII(SEQ ID NO:7) (5′-TGACCCACGCTCGCC) and VIII (SEQ ID NO:8)(3′-ACTGGGTGCGAGCGGGCCTCTATTTAATACA) in water, heating at 95° C. for 5min and slow cooling to room temperature. Labeled duplexes were preparedby mixing DNA duplex (400 nM), dATP, dGTP and dTTP (33 μM of each),[α-33P]CTP (1.5 μM, Hartmann Analytic, Germany) and Klenow Fragment(0.16 u/μL, Fermentas Life Sciences) and incubating in Klenow reactionbuffer at 37° C. for 30 min following incubation at 75° C. for 15 min.20-100 nM DNA duplexes were then treated with 125 nM M.HhaI, 1000 nMM.HpaII, or 120 nM M.SssI in buffer (5-20 μL, 50 mM MOPS, 50 mM MES pH7.0 (for M.HhaI) and pH 7.5 (for other methyltransferases), 1 mMNa₂EDTA, 15 mM NaCl, 0.2 mg/ml bovine serum albumin, 5% glycerol) and 13mM formaldehyde for 1 hour. Modification removal reactions wereperformed by incubating a 20 μM duplex oligodeoxyribonucleotides with acognate DNA methyltransferase (2 μM M.HhaI, 2 μM M.HpaII or 1.2 μMM.SssI) for 2-5 hours at 37° C. in buffer (10 mM Tris-HCl pH 7.4, 50 mMNaCl, 0.5 mM Na₂EDTA 0.2 mg/ml bovine serum albumin, 5% glycerol). Forlabeled nucleotide analysis, DNA was precipitated with 3 volume ofethanol, dissolved in nuclease BAL31 buffer (5 μL) with nuclease BAL31(0.4 u) (Fermentas Life Sciences) and incubated for 1 hours at 30° C.0.5-3 μl aliquotes were spotted on TLC plates (PEI CelluloseF, 20×20 cm,Merck). TLC plates were eluted with isobutyric acid/water/conc. ammonia,(66:17:4, vol/vol/vol). Plates were dried overnight and radioactivebands were autoradiographed to an imaging plate (Fujifilm, Japan)followed by scanning with a FLA-5100 phosphoimager. Radioactive spotswere quantitated using MultiGauge software (Fujifilm). Modified2′-deoxy-5′-mononucleotides (dXMP) were detected as radioactive spots inaddition to the major spot of 2′-deoxycytosine-5′-monophosphate (dCMP).The position of a modified nucleotide (X) relative to that of theunmodified C nucleotide (dCMP) (Rc(X)═R_(f)(dXMP)/R_(f)(dCMP)) wasdetermined and was used for its chromatographic identification.

Example 2

Target nucleotide analysis in a hmC-containing duplexoligodeoxyribonucleotide after treatment with the HhaI DNA cytosine-5methyltransferase (M.HhaI).

TLC analysis of modification products obtained after treatment ofhmC-containing DNA duplex with M.HhaI indicated a decrease of the ratioof hmC and C nucleotides (Rc values of 0.85 and 1.0) (see FIG. 2, lanes1 and 2). Control reaction (lane 3) contained catalytically inactivemutant (C81S) of M.HhaI. Thus the 5-hydroxymethyl group is removed fromthe target hmC residue yielding unmodified cytosine in the presence ofcatalytically active M.HhaI.

Example 3

Target nucleotide analysis in duplex oligodeoxyribonucleotidescontaining hmC, C or 5mC at the target position after treatment withL-cysteine and the HhaI DNA cytosine-5 methyltransferase (M.HhaI).

20 nM duplex oligodeoxyribonucleotides V:VI (cognate-C), V:VI-methylated(cognate-5mC), V:VI-hydroxymethylated (cognate-hmC), VII:VIII(nonspecific-C) or VII:VIII-hydroxymethylated (nonspecific-hmC) (FIG. 3,lanes 1-5, respectively) was incubated with 50 mM L-cysteine and M.HhaI(120 nM) for 1 hour at room temperature. An additional labeled spot(R_(C)=0.55) corresponding to coupling product Cys-hmC was observed(lane 3) only when the cognate-hydroxymethylated DNA was used as asubstrate. Thus M.HhaI adds exogenous nucleophiles in asequence-specific manner to hmC residues, but not to C or 5mC residues.

Example 4

Target nucleotide analysis in a hmC-containing duplexoligodeoxyribonucleotide after treatment with L-selenocysteine and theDNA cytosine-5 methyltransferase (M.HhaI).

TLC analysis of modification products obtained after treatment ofhmC-containing cognate DNA duplex with L-selenocysteine and M.HhaIindicated the appearance of a new modified nucleotide SeCys-hmC (Rcvalue of 0.6; FIG. 3, compare lanes 6 and 7). Thus L-selenocysteine iscoupled to the target 5-hydroxymethylcytosine residue in DNA in thepresence of M.HhaI.

Example 5

Target nucleotide analysis in a hmC-containing duplexoligodeoxyribonucleotide after treatment with the SssI DNA cytosine-5methyltransferase (M.SssI).

TLC analysis of modification products obtained after treatment ofhmC-containing DNA duplex with M.SssI indicated a decrease of the ratioof hmC and C nucleotides (FIG. 2, compare lanes 7-8). Control reaction(lane 9) contained thermally inactivated M.SssI. Thus the5-hydroxymethyl group is removed from the target hmC residue yieldingunmodified cytosine in the presence of catalytically active M.SssI.

Example 6

Target nucleotide analysis in a hmC-containing duplexoligodeoxyribonucleotide after treatment with the HpaII DNA cytosine-5methyltransferase (M.HpaII).

TLC analysis of modification products obtained after treatment of ahmC-containing DNA duplex with M.HpaII indicated a decrease of the ratioof hmC and C nucleotides (FIG. 2, compare lanes 4-5). Control reaction(lane 6) contained thermally inactivated M.HpaII. Thus the5-hydroxymethyl group is removed from the target hmC residue yieldingunmodified cytosine residue in the presence of catalytically activeM.HpaII.

Examples 7-11

Sequence-specific modifications of large DNA molecules containing5-hydroxyalkyled target cytosine residues with a DNA cytosine-C5methyltransferase.

Sequence-specific modifications by DNA cytosine-05 methyltransferasesHhaI, SssI and HpaII were investigated using a DNA protection assay.This assay makes use of the fact that DNA methyltransferase-catalyzedmodifications of nucleobases within the recognition sequence ofrestriction endonucleases can protect the DNA against fragmentation ofthese enzymes. DNA containing unmodified target sites of a restrictionendonuclease is readily fragmented by the restriction endonuclease,whereas covalent modification of the target sites blocks the DNAcleavage. Occurrence of fragmentation is then analyzed by agarose gelelectrophoresis.

A 618 bp fragment of plasmid pUC18 (pUC-618), which contains a singletarget site for M.HhaI, 2 sites for HpaII and 32 sites for M.SssI, wasused as the DNA substrate. pUC-618 was prepared by PCR amplification ofthe pUC19 template (Fermentas Life Sciences) using Dir(5′-AACGTTGTTGCCATTGCTAC) (SEQ ID No:11) and Rev(5′-GCTCATGAGACAATAACCCTGA) (SEQ ID No:12) primers and Taq DNAPolymerase (Fermentas Life Sciences). The PCR fragment was purified bySephacryl S-400 (GE Healthcare) following the precipitation by ethanol.

hmC modification at the GCGC target site was introduced by treatment of100 nM pUC-618 with 50 nM M.HhaI in buffer (50 mM MOPS, 50 mM MES pH7.0, 1 mM Na₂EDTA, 15 mM NaCl, 0.2 mg/ml bovine serum albumin, 5%glycerol) with 13 mM formaldehyde for 1 hour at room temperature.5-hydroxyethylcytosine (heC) modification at the GCGC target site wasintroduced by treatment of 100 nM pUC-618 with 50 nM M.HhaI with 800 mMacetaldehyde for 1 hour at room temperature as above. hmC modificationat the CG target sites was introduced by treatment of 200 nM pUC-618with 1200 nM M.SssI (Q142A/N370A) and 13 mM formaldehyde for 1 hour atroom temperature as described above. hmC modification at the target CCGGsites was introduced by treatment of 200 nM pUC-618 with 2000 nM M.HpaIIand 13 mM formaldehyde for 1 hour at room temperature as describedabove. Reactions were stopped by heating at 75° C. for 20 min. DNA wasprecipitated by 3 volumes of ethanol and washed ones by 75% of etanol.Modification removal reactions were performed by incubating a duplexoligodeoxyribonucleotides with a cognate DNA methyltransferase for 2-5hours at 37° C. in buffer (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 0.5 mMNa₂EDTA 0.2 mg/ml bovine serum albumin, 5% glycerol).

DNA cleavage with restriction endonucleases was performed according tomanufacturer's recommendations (Fermentas Life Sciences). Samples weresupplemented with ⅙ of 6× Loading Dye Solution and analyzed by 2%agarose gel electrophoresis.

Fluorescent labeling of DNA fragments was achieved by treatment ofhmC-containing pUC-618 fragment (13 μL, 0.2 μM) with 25 mM 1,4dithiothreitol in the presence of M.HhaI (1 μM) or M.SssI (6.5 μM) for 1hour at room temperature. DNA was then purified using Qiagen NucleotideRemoval Kit and treated with 1 mM Oregon Green 488 maleimide(Invitrogen) (dissolved in dimethylformamide, Fluka) for 12 hour at roomtemperature in the dark in buffer (20 μL, 100 mM Tris-HCl pH 7.4, 5 mMTCEP). DNA was purified with Nucleotide Removal Kit (Qiagen) andanalyzed by 2% agarose gel electrophoresis (10 V/cm) in the absence ofethidium bromide. Gels were first scanned with a Fuji FLA-5100 imagingsystem using a 473 nm laser and then inspected in a UV-imager afterstaining with ethidium bromide.

Example 7

Sequence-specific modification of a 618 bp hmC-containing DNA fragmentin the presence of M.HhaI.

FIG. 4 (lanes 1-4) shows that the hmC modification makes the pUC-618fragment resistant to R.Hin6I (lanes 1 and 2). Treatment with wild typeM.HhaI renders the GCGC site largely cleavable with R.Hin6I (lane 3),however treatment with the catalytically impaired mutant C81S ofM.HhaI—does not (lane 4). Thus catalytic action of M.HhaI leads toefficient removal of 5-hydroxymethyl groups from the GCGC target site ina DNA fragment.

Example 8

Sequence-specific modification of a 618 bp5-hydroxyethylcytosine-containing DNA fragment in the presence ofM.HhaI.

FIG. 4 (lanes 5-8) shows that the heC modification makes the pUC-618fragment resistant to R.Hin6I cleavage (compare lanes 5 and 6).Treatment with wild type M.HhaI renders the GCGC site largely cleavablewith R.Hin6I, (lane 7), however treatment with the catalyticallyimpaired mutant C81S of M.HhaI—does not (lane 8). Thus catalytic actionof M.HhaI leads to removal of 5-hydroxyethyl groups from the GCGC targetsite in a DNA fragment.

Example 9

Sequence-specific modification of a 618 bp hmC-containing DNA fragmentin the presence of M.SssI.

FIG. 5 shows that the hmC modification makes the pUC-618 fragmentpartially resistant to R.Hin6I cleavage (lane 1). Treatment with M.SssIrenders the GCGC site more cleavable with R.Hin6I (increased amount ofcleavage products in lane 2). Thus action of M.SssI leads to removal of5-hydroxymethyl groups from the GCGC target site in a DNA fragment.

Example 10

Sequence-specific modification of a 618 bp hmC-containing DNA fragmentin the presence of M.HpaII.

FIG. 5 shows that the hmC modification makes the pUC-618 fragmentpartially resistant to R.HpaII (lane 3). Treatment with M.HpaII rendersthe CCGG site more cleavable with R.HpaII (increased amount of cleavageproducts in lane 4). Thus action of M.HpaII leads to removal of5-hydroxymethyl groups from the CCGG target site in a DNA fragment.

Example 11

Sequence-specific conversion hmC residues to cytosines in human genomicDNA using a DNA cytosine-5 methyltransferase, M.SssI.

90 ng of human genomic DNA (gDNA) (isolated from post mortem humanbrains) was incubated in 25 μl buffer (50 mM MOPS, 50 mM MES pH 7.5, 1mM Na₂EDTA, 15 mM NaCl, 0.2 mg/ml bovine serum albumin, 5% glycerol)with 1200 nM M.SssI overnight at 37° C. DNA was purified using QiagenNucleotide Removal Kit and fragmented with R.MspI or R.HpaII (20 u)(Fermentas Life Science) for 3 hours at 37° C. Then RnaseA (5 μg) andFastAP (0.5 u) was added and incubation was continued for one more hour.

DNA again was purified with Qiagen Nucleotide Removal Kit and labeledusing T4 Polynucleotide Kinase (Fermentas Life Science) and [γ-³³]ATP(Hartmann Analytic). Then DNA was precipitated with 3 volumes of ethanoland digested with Lambda Exonuclease (5 u) for one hour at 37° C. 0.5-3μl aliquotes were spotted on TLC plates (PEI CelluloseF, 20×20 cm,Merck). TLC plates were eluted with isobutyric acid/water/conc. ammonia,(66:17:4, vol/vol/vol). Plates were dried overnight and radioactivebands were autoradiographed to an imaging plate (Fujifilm, Japan)followed by scanning with a FLA-5100 phosphoimager. Densitometricanalysis of autoradiographic images was performed using MultiGaugesoftware (Fujifilm). FIG. 7 shows a clear presence of hmC and 5mCresidues at the second residue of CCGG sites (underlined) in genomic DNAfrom human brain—hmC and 5mC containing sites are cleaved and labeled atthe second nucleotide with R.MspI (see lane 2 and corresponding blackdensity trace on right) but are not cleaved and thus not labeled (lane 1and dotted density trace on right) upon treatment with R.HpaII(Kriaucionis, S. & Heintz, N. Science published online,doi:10.1126/science.1169786). Treatment of genomic DNA with M.SssIreduces the amount of detectable hmC at the CCGG (lane 3 andcorresponding gray trace on right). The right side arrow points at theposition of hmC in the density traces. Thus the 5-hydroxymethyl group isremoved from the target cytosine residues at CCGG cites in human genomicDNA in the presence of M.SssI.

Example 12

Sequence-specific fluorescent labeling of a 618 bp hmC-containing DNAfragment 1,4-dithiothreitol (DTT) in the presence of M.HhaI or M.SssI.

FIG. 7 shows that treatment with DTT in the presence of M.HhaI or M.SssI(lanes 3 and 2, respectively) followed by thiol-specific ligation withOregon Green 488 maleimide renders the pUC-618 fragment fluorescent uponillumination with a 473 nm light. Thus labeling of a hmC-containingfragment can be achieved by DNA methyltransferase-directed coupling ofDTT followed by chemo-selective ligation with a maleimide-reactivefluorescent label. A control reaction carried out without a MTase showsno visible DNA fragments in the absence of ethidium bromide staining.

In the following Examples 13 to 20 the alpha-hydroxylated residues areproduced by reaction of an oligodeoxyribonucleotide with an aldehyde.

Example 13

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and 2-mercaptoethanol inthe presence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and 300 mM 2-mercaptoethanol (Q=S; LX═CH₂CH₂OH) revealed in addition tothe natural nucleosides dC, dG, dT and dA two new products: one with theretention time of 5-hydroxymethyl-2′-deoxycytidine and another with aretention time of 16.8 min (elution buffer 20 mM ammonium acetate pH5.5). This new product was analyzed by coupled ESI-MS (m/z: 340 [M+Na]⁺,224 [5-(2-hydroxyethyl)thiomethylcytosine+Na]⁺) and separately by HR-MS(m/z found: 318.1118; calculated for [M+H]+C₁₂H₂₀N₃O₅S; 318.1119). Theobserved masses are in agreement with5-(2-hydroxyethylthio)methyl-2′-deoxycytidine. Thus 2-mercaptoethanol iscoupled to a 5-hydroxymethylcytosine residue in DNA by M.HhaI. See FIG.8, trace 3.

Example 14

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and L-cysteine in thepresence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 50 mM L-cysteine (Q=S; LX═CH₂CH(CO₂H)NH₂) in the presenceof M.HhaI revealed in addition to the natural nucleosides dC, dG, dT anddA two new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine (Example 1) and another with aretention time of 2.7 min (elution buffer 20 mM ammonium acetate pH5.5). This new products were analyzed by HR-MS (m/z found: 361.1176;calculated for [M+H]+C₁₃H₂₁N₄O₆S: 361.1176). The observed mass is inagreement with 5-(S-cysteinyl)methyl-2′-deoxycytidine. Thus L-cysteineis coupled to a 5-hydroxymethylcytosine residue in DNA by M.HhaI. SeeFIG. 8, trace 4.

Example 15

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and5′-thio-5′-deoxyadenosine in the presence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 400 μM 5′-thio-5′-deoxyadenosine (Q=S;LX=5′-deoxyadenosine) in the presence of M.HhaI revealed in addition tothe natural nucleosides dC, dG, dT and dA two new products: one with theretention time of 5-hydroxymethyl-2′-deoxycytidine (Example 1) andanother with a retention time of 27.5 min (elution buffer 20 mM ammoniumacetate pH 5.5).

This new product was analyzed by coupled ESI-MS (m/z: 545 [M+Na]+, 429[5-(5′-denosyl)thiomethyl-2′-deoxycytidine+Na]+) and separately by HR-MS(m/z found: 523.1718; calculated for [M+H]+C₂₀H₂₇N₈O₇S: 523.1718). Theobserved masses are in agreement with5-(5′-adenosyl)thiomethyl-2′-deoxycytidine. Thus5′-thio-5′-deoxyadenosine is coupled to a 5-hydroxymethylcytosineresidue in DNA by M.HhaI. See FIG. 8, trace 5.

Example 16

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and cysteamine in thepresence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 12 mM cysteamine (Q=S; LX═CH₂CH₂NH₂) in the presence ofM.HhaI revealed in addition to the natural nucleosides dC, dG, dT and dAtwo new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine (Example 1) and another with aretention time of 4.0 min (elution buffer 20 mM ammonium acetate pH5.5).

This new product was analyzed by coupled ESI-MS (m/z: 339 [M+Na]+, 223[5-(2-aminoethyl)thiomethylcytosine+Na]+). The observed masses are inagreement with 5-(2-aminoethyl)thiomethyl-2′-deoxycytidine. Thuscysteamine is coupled to a 5-hydroxymethylcytosine residue in DNA byM.HhaI. See FIG. 8, trace 6.

Example 17

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and 1,4-dithiothreitol(1,4-dithio-2,3-dihydroxy-butane) in the presence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 50 mM 1,4-dithiothreitol (Q=S; LX═CH₂CH(OH)CH(OH)CH₂SH) inthe presence of M.HhaI revealed in addition to the natural nucleosidesdC, dG, dT and dA two new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine (Example 1) and another with aretention time of 22.1 min and 22.8 min (two isomers) (elution buffer 20mM ammonium acetate pH 5.5).

This new product was analyzed by coupled ESI-MS (m/z: 416 [M+Na]+). Theobserved masses are in agreement with5-(2,3-dihydroxy-4-mercaptobutyl)thiomethyl-2′-deoxycytidine. Thus1,4-dithiothreitol is coupled to a 5-hydroxymethylcytosine residue inDNA by M.HhaI. See FIG. 8, trace 7.

Example 18

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and selenocysteine in thepresence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 50 mM selenocysteine (Q=Se; LX═CH₂CH(CO₂H)NH₂) in thepresence of M.HhaI revealed in addition to the natural nucleosides dC,dG, dT and dA two new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine and another with a retention time of3.1 min (elution buffer 20 mM ammonium acetate pH 5.5). This new productwas analyzed by HR-MS (m/z found: 409.0621, calculated for[M+H]+C₁₃H₂₁N₄O₆Se: 409.0621). The observed mass is in agreement with5-(2-amino-2-carboxyethyl)selenomethyl-2′-deoxycytidine. Thusselenocysteine is coupled to a 5-hydroxymethylcytosine residue in DNA byM.HhaI. See FIG. 8, trace 8.

Example 19

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and selenocysteamine in thepresence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 1,2 mM selenocysteamine (Q=Se; LX═CH₂CH₂NH₂) in thepresence of M.HhaI revealed in addition to the natural nucleosides dC,dG, dT and dA two new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine and another with a retention time of5.0 min (elution buffer 20 mM ammonium acetate pH 5.5).

This new product was analyzed by coupled ESI-MS (m/z: 386 [M+Na]+). Theobserved masses are in agreement with5-(2-aminoethyl)selenomethyl-2′-deoxycytidine. Thus selenocysteamine iscoupled to a 5-hydroxymethylcytosine residue in DNA by M.HhaI. See FIG.8, trace 9.

Example 20

Nucleoside composition analysis of a duplex oligodeoxyribonucleotideafter sequential treatment with formaldehyde and hydroxylamine in thepresence of M.HhaI.

HPLC analysis of nucleoside composition of the modified duplexoligodeoxyribonucleotide (I:II) treated with 13 mM formaldehyde (R═H)and then with 50 mM hydroxylamine (Q=NH; LX═OH) in the presence ofM.HhaI revealed in addition to the natural nucleosides dC, dG, dT and dAtwo new products: one with the retention time of5-hydroxymethyl-2′-deoxycytidine and another with a retention time of5.0 min (elution buffer 20 mM ammonium acetate pH 5.5) in agreement withthe formation of 5-hydroxylaminomethyl-2′-deoxycytidine. Thushydroxylamine is coupled to a 5-hydroxymethylcytosine residue in DNA byM.HhaI. See FIG. 8, trace 10.

1. A method of using a cofactor-free methyltransferase for targetedconversion of a modified biomolecule, the method comprising removing ata methyltransferase target site a modifying moiety of formula —CH(OH)—R,wherein R is hydrogen or C₁-C₁₂-alkyl, from the modified biomoleculeusing a cofactor-free methyltransferase resulting in targeted conversionof the modified biomolecule into an unmodified biomolecule.
 2. A methodfor targeted conversion of a modified biomolecule, comprising incubatingthe modified biomolecule bearing a modifying moiety of formula—CH(OH)—R, wherein R is hydrogen or C₁-C₁₂-alkyl, with a cofactor-freedirecting methyltransferase under conditions compatible with enzymaticactivity of the methyltransferase, and obtaining targeted conversionresulting from: i) covalent removal of said modifying moiety at thetarget site; or ii) derivatization of said modifying moiety at thetarget site by covalent coupling of non-cofactor nucleophiliccompound(s) of general formula HQ-LX, wherein X represents a functionalgroup or a reporter group attached via a linker L, and Q is selectedfrom S, Se, O, N, or C.
 3. The method of claim 1 or claim 2 wherein saidbiomolecule is a nucleic acid molecule, and said methyltransferase is aDNA cytosine-5 methyltransferase selected from the group consisting ofM.HhaI, M.SssI, and M.HpaII or derivatives thereof.
 4. The method ofclaim 2, wherein the modified biomolecule is a naturally or artificiallymodified biomolecule, and wherein R is hydrogen or —CH₃ and Q is S orSe.
 5. The method of claim 2 further comprising directly or subsequentlyincorporating a reporter group suitable as a label and allowingidentification of the labeled molecule among other unlabeled molecules.6. The method of claim 5, wherein the label is selected fromfluorophores, fluorescence quenchers, chromophores, affinity tags,stable paramagnetic groups, groups containing radioactive or stable rareisotopes, groups containing heavy atoms suitable for phasing X-raydiffraction data, crosslinking agents, nucleic acids cleaving groups,haptens, nanoparticles, beads and combinations thereof.
 7. A method fordetecting hydroxymethylated target sites in a biomolecule, comprisingderivatizing or labeling the biomolecule by coupling non-cofactornucleophilic compounds of general formula HQ-LX, wherein X represents afunctional group or a reporter group attached via a linker L, and Q isselected from S, Se, O, N, or C in the presence of a cofactor-freemethyltransferase, and detecting whether the target sites of saidmethyltransferase have been modified, wherein modification of the targetsite of said methyltransferase is indicative of the presence ofhydroxymethylated target site.
 8. The method of claim 7, wherein thecoupled compound interferes with nucleic acid amplification at therecognition sites of the methyltransferase; and hydroxymethylated targetsites are detected by testing whether amplification of the nucleic acidmolecule at the recognition sites of the methyltransferase has beenretarded.
 9. The method of claim 7, wherein the coupled compoundcomprises a fluorescent label; and hydroxymethylated target sites aredetected by measuring the presence or amount of fluorescence in saidnucleic acid molecule.
 10. The method of claim 7, wherein the coupledcompound is added to a 5-hydroxymethylcytosine residue in DNA and cannotbe added to a 5-methylcytosine or a cytosine residue.
 11. The method ofclaim 5, wherein the coupled compound or a label in a derivatizedbiomolecule is identified by DNA sequencing, hybridization, massspectrometry or analysis of nucleoside composition by enzymaticfragmentation and chromatography.
 12. A kit comprising a cofactor-freedirecting methyltransferase or a cofactor-free directingmethyltransferase and non-cofactor nucleophilic compound(s) in separatecontainers, and instructions for use to perform targeted conversion of amodified biomolecule or to detect hydroxymethylated target sites in abiomolecule.
 13. The kit of claim 12, further comprising scavengingcompound(s) in separate containers.
 14. A 2′-deoxycytidine derivativeformed in DNA, which is 5-(Se-selenocysteinyl)methyl-2′-deoxycytidine.15. The method of claim 1 wherein R in the modifying moiety beingremoved is hydrogen.
 16. The method of claim 1 wherein R in themodifying moiety being removed is a lower alkyl.
 17. The method of claim1 wherein R in the modifying moiety being removed is —CH₃.
 18. Themethod of claim 2 wherein R in the modifying moiety is hydrogen.
 19. Themethod of claim 2 wherein R in the modifying moiety is a lower alkyl.20. The method of claim 2 wherein R in the modifying moiety is —CH₃. 21.The method of claim 3 wherein the nucleic acid molecule is DNA.
 22. Themethod of claim 7 wherein the coupled compound or a label in aderivatized biomolecule is identified by DNA sequencing, hybridization,mass spectrometry or analysis of nucleoside composition by enzymaticfragmentation and chromatography.
 23. The method of claim 5 or claim 22wherein the derivatized biomolecule is DNA.